PROJECT SUMMARY The objective of the proposed research is to determine the mechanisms by which JC polyomavirus (JCPyV) interacts with cellular receptors to invade host cells and cause infection. JCPyV infects the majority of the human population and establishes a lifelong, asymptomatic infection in the kidney. In immunocompromised individuals, JCPyV can spread to the central nervous system (CNS) and cause a lytic infection in glial cells, resulting in the fatal, demyelinating disease progressive multifocal leukoencephalopathy (PML). Individuals with HIV and those receiving immunomodulatory therapies for diseases such as multiple sclerosis are at heightened risk for PML development. PML can be fatal within three months to two years of symptom onset as there are no effective treatments for this devastating disease. The mechanisms by which JCPyV engages cellular receptors to activate endocytic and signaling pathways for infection are poorly understood. Virus interactions with host cell receptors are the initial steps in the infectious viral replication cycle and are thus critical regulators of host cell susceptibility and disease pathogenesis. Therefore, to better define mechanisms of JCPyV host cell invasion, infection, and viral disease, it is imperative to gain a detailed understanding of the interactions between JCPyV and host cell receptors. Our previous work elucidated that ?2,6-linked lactoseries tetrasaccharide c (LSTc) is the primary attachment receptor and JCPyV entry is mediated by serotonin 5-hydroxytryptamine subtype 2 receptors (5-HT2Rs). However, the mechanism by which the 5-HT2Rs mediate viral internalization is unknown. Our preliminary data demonstrates that JCPyV entry by 5-HT2Rs is facilitated by clathrin-mediated endocytosis in a ?-arrestin dependent manner. Interestingly, endocytosis of 5-HT2Rs and accumulation of ?-arrestin in endocytic vesicles can result in the activation of the MAPK pathway, which we have also shown to be essential for JCPyV infection. We hypothesize that JCPyV utilizes the 5-HT2Rs to mediate viral entry but also to activate the MAPK pathway to reprogram host cells to drive viral infection. Three integrated specific aims are proposed to understand the mechanisms by which JCPyV interacts with 5-HT2Rs to mediate internalization into host cells and usurp cellular signaling networks to drive viral infection. Findings from this research will enhance our understanding of how viral entry and signaling events promote viral infection. This work will also elucidate novel viral and cellular factors that could be targeted for the future development of effective antiviral therapies for JCPyV as well as other viruses. Additionally, this research will contribute to a broader understanding of serotonin receptor biology, G-protein coupled receptor signaling, and demyelinating disorders of the CNS.